The basic steps of Western Blot technology are to isolate protein samples based on protein molecular weight, gel SDS-PAGE electrophoresis, identify target protein or specific protein phosphorylation site by protein specific antibody, and finally obtain the target protein expression in samples by analyzing antigen antibody binding location and signal intensity.
| STEP | DETAIL |
| protein extraction | A.The experimental subjects were tissue samples. A suitable amount of fresh tissue samples (250-500 mg) were taken, and 1 ml of total protein extraction reagent (or nucleoprotein extraction reagent) containing protease inhibitor was added. The total protein was extracted after homogenization. B.Cell samples were taken from each sample. Cells were washed by PBS. Total protein extraction reagents (or nucleoprotein extraction reagents) containing protease inhibitors (0.1ml~1ml) were added to PBS to extract total protein. |
| protein quantification | The sample concentration was determined according to the operation instructions of KCTMBCA protein quantitative kit. |
| discontinuous polyacrylamide gel electrophoresis(SDS-PAGE) | The prepared sample solution and the protein molecular weight standard labeled by biotin were sampled separately. The standard was added to the first pore and the protein was separated by electrophoresis. |
| protein transfer to nitrate fiber membrane or PVDF membrane | |
| closure of membranes and incubation of antibodies | A.The membrane was incubated at room temperature for 1 hour in 5% BSA solution to seal the nonspecific binding of the membrane. B.The blocked membrane was incubated at room temperature for 1.5 hours with the first antibody, and the antigen and antibody were bound. C.The second antibody labeled with HRP was added to bind the first antibody and the antibiotic antibody labeled with HRP to bind the molecular weight standard. The membrane was incubated at room temperature for 1 hour. GAPDH antibody labeled with HRP can simultaneously detect GAPDH content. |
| result detection | Chemiluminescence detection, film and chemiluminescence substrate incubation, X-ray film exposure and development. The image is scanned and saved as a computer file, and the gray value of each special strip on the image is digitized by ImageJ analysis software. |
| data analysis | The gray value of the target protein is divided by the gray value of internal reference GAPDF to correct the error. The result represents the relative content of the target protein in a sample. |
| provide experimental reports | Detailed experimental methods and Western Blot experimental results are included. |
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